Dendritic nanogel for drug delivery platform

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DEGREE PROJECT IN CHEMICAL SCIENCE AND ENGINEERING, SECOND CYCLE, 30 CREDITS STOCKHOLM, SWEDEN 2018

Dendritic nanogel for drug delivery

platform

GÜNES UYSAL

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Supervisor:

Yuning Zhang, KTH

Co-supervisor:

Oliver Andrén, KTH

Examinator:

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Acknowledgments

Firstly, I would like to thank Professor Michael Malkoch for giving me the opportunity to work with this interesting project.

In particular, I would like to thank my two amazing supervisors Oliver Andrén and Yuning Zhang for their support and valuable input throughout the project.

I am also very thankful that I did my master thesis at Ytgruppen, which is a very positive and supportive group.

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List of abbreviations

CDI 1,1-Carbonyldiimidazole

Bis-MPA 2,2-bis (hydroxymethyl)propionic acid

mPEG Poly (ethylene glycol) monomethyl ether (mPEG)

pTSA p-Toluenesulfonic acid

CDCl3 Chloroform-D

CHCl3 Chloroform

hb mPEG-nk-Gm-OH The n stands for the molecular weight of mPEG (2k= 2000 g/mol and 5k= 5000 g/mol). m stands for the generation (2-4), OH functionality at the periphery. hb stands for hyperbranched.

hb mPEG-nk-Gm-allyl The n stands for the molecular weight of mPEG (2k= 2000 g/mol and 5k= 5000 g/mol). m stands the generation (2-4), allyl

functionality at the periphery. hb stands for hyperbranched.

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Abstract

The development of nano- based drug carriers is of high importance in anti-cancer treatment as anticancer drugs suffers from limitations as low aqueous solubility, non-selective targeting, off-target degradation and low therapeutic concentrations at target site. Hyperbranched polymers are potential candidates as drug carrier due to its unique properties as globular shape, high number of functional groups and high degree of branching. In addition, hyperbranched polymers are synthesized via one-step polymerization reaction with high yields, low costs and good scale-up possibilities. In this project a library of hyperbranched linear-dendritic hybrid materials based of 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and monofunctional poly (ethylene glycol) (mPEG) was synthesized via the Fischer esterification reaction. The materials were then post functionalised with hydrophobic allyl groups. The materials assembled into micelles in water and candidates with best self-assembly ability were used to fabricate dendritic nanogels by UV-induced cross-linking. The formed dendritic nanogels obtained a hydrodynamic volume between 124-200 nm, which indicates that these dendritic nanogels can be used as drug carrier and accumulate at target-site via the enhanced permeability and retention (EPR) effect. The dendritic nanogels inner core was also successfully attached with cationic, hydrophobic and anionic groups respectively. This confirmed that the dendritic nanogels have the potential to encapsulate different types of cargo such as DNA or hydrophobic drugs in the inner core.

KEYWORDS: hyperbranched polymers, anticancer treatment, dendritic nanogels, EPR-effect,

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Sammanfattning

Utveckling av polymer baserade läkemedelsbärare i nanostorlek har blivit allt viktigare för att effektivisera behandling och diagnosering av olika sjukdomar, speciellt cancer. Flera läkemedel som används i kemoterapi har bristfälliga egenskaper som låg löslighet i vatten, oönskad nedbrytbara till dess inaktiva form, och distribution i stora volymer till oönskade organ p.g.a. dess icke-selektiva förmåga. Nanopartiklar är små partiklar med diameter 1-500 nm som genom passiv/aktiv transport kan passera olika biologiska barriärer och transportera läkemedel i optimala mängder till specifika celler. Denna selektiva transport bidrar till ökad terapeutiskt index och minskning av toxiska effekter i övriga delar av kroppen. Hyperförgrenade linjär-dendritiska hybrider är en subgrupp av dendritiska polymer som har stor potential att användas som byggstenar i utvecklingen av läkemedelsbärare. I detta projekt producerades ett bibliotek av hyperförgrenade linjär-dendritiska material via Fischer esterifikation reaktionen som är en snabb, billig och uppskalningsbar produktionsmetod. Vidare post funktionaliserades materialen med allyl grupper för produktion av nano geler genom UV-inducerad korslänkning och vidare funktionalisering. Samtliga producerade hyperförgrenade linjär-dendritiska material hade förmågan att bilda miceller i vatten. Materialen med bäst micelle bildningsförmåga användes för att kemiskt korslänka dem och producera nano geler. Nano gelernas inre del funktionaliserades framgångsrikt med tre olika funktionella grupper; katjoniska, anjoniska och hydrofoba via resterande fria allyler. Detta påvisar att dessa dendritiska nano geler har potential att bära olika material som hydrofobiska läkemedel eller genetiskt material. Dom producerade nano gelerna hade en hydrodynamisk volym inom intervallet 124-200 nm. Detta är fördelaktigt då dem kan transporteras till tumörområdet via ökad permeabilitet och retention, också kallad EPR effekten, utan att initiera ett immunologiskt svar eller filtreras från blodomloppet via njuren.

SÖKORD: nano partiklar, anti-cancer behandling, hyperförgrenade linjär-dendritiska hybrider

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1. Introduction

Cancer is a group of diseases characteristic for its abnormal proliferation of cells. This is because of genetic alterations, also called mutations, in essential genes regulating important cellular mechanisms such as DNA repair or apoptosis.[1, 2] _{Due to limitations in diagnosis and} treatment cancer has become a disease with high morbidity and mortality world-wide.[3, 4] Cancer is mainly treated by surgery, chemotherapy, radiation therapy, or, a combination depending on progression of disease. Chemotherapy currently used faces two important challenges; the therapeutic drugs inability to differentiate between cancer cells and normal cells and the low therapeutic index. The non-selective delivery of anticancer drugs, high administration doses and broad distribution to non-target organs cause systematic toxicity and adverse effect for the patient.[5, 6] _{Other drawbacks with anticancer drugs, which} decreases the therapeutic efficiency, are their low aqueous solubility, low molecular weight, and their degradation before reaching site of disease.[7]

Advances in nanotechnology have driven scientists around the world to develop polymeric nano-based carriers with the ability to protect the cargo from inactivation and degradation, deliver it safely to a target site with minimum distribution to non-target organs, and release it in optimal concentrations at site of disease. Different polymeric materials have been studied as material to develop drug delivery systems (DDS) such as micelles, liposomes and nanoparticles to improve the therapeutic efficiency of many therapeutic drugs and minimize systematic toxicity. [6, 8]_{Other examples of materials for drug delivery are metallic NPs and} inorganic NPs. Polymers are the ideal material for drug delivery systems due to its ease of manufacture, tuneable physicochemical properties (compositions, hydrophobicity/hydrophilicity, crystallinity), variety in size, possibility to control degradation kinetics and their ability to escape immunological response.[9, 10]_{Another advantage is its} biodegradability compared with metallic NPs, where metal release and accumulation is an issue.[11]

Nanoparticles are highly suitable as DDS due to its beneficial properties as small size, high surface to ratio, and presence of functional groups which can be easily functionalised. The ease of chemical modifications gives the ability to create target specific nanoparticles by the attachment of biological moieties as antibodies, carbohydrates and peptides. Nanoparticles are interesting in drug delivery due to its ability to increase the therapeutic index, increase the circulation time of drugs, reduce the frequency of administration of drugs and contribute with a controlled release of drugs. Nanoparticles can besides drug delivery be used in biomedical applications such as gene delivery and diagnostic imaging. [8, 12-14]

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2. Background

2.1 Drug delivery systems (DDS)

A drug delivery system (DDS) can be a polymeric device, which shields the capsulated therapeutic agent from the environment (e.g. hydrolysis or interaction with tissue components) and transports it to the site of disease in the body, either by passive or active targeting. The usage of a carrier increases the accumulation of the drug at target site by selective transport in contrast to free drug, while minimizing adverse toxic effects on off-target tissues. Additionally, it maintains the stability of the therapeutic drug. This results in increased therapeutic index.[4, 15]

Polymers can be designed as different types of DDSs structures such as micelles, polymer- liposomes hybrids, nanogels and dendrimers. DDSs can be used in biomedical applications such as drug delivery, gene delivery and diagnostic imaging.[6, 16]

2.2 Passive targeting

Passive targeting is the fundamental of DDS assisted cancer targeting. A DDS with size from 10-200 nm can be accumulated to the solid tumour (e.g. tumour tissue) via the permeability and retention effect.[17] _{In contrast to healthy tissue is the vasculature close to tumour tissue} abnormal at a microscopic level (see figure 1). The leaky vasculature and impaired lymphatic drainage associated with tumour tissue allows nanoparticles to pass through the gaps between the endothelial cells of the blood vessel wall and accumulate at tumour site.[6, 18-20]_In order to penetrate by the EPR effect the nanoparticle needs to have a size between 10-200 nm. A nanoparticle needs to be bigger than 10 nm in size to avoid clearance from the body via the kidney and a size below 200 nm to avoid response by the immune system.[15, 21] In this project, we focused on generating a DDS with preferable size to enable EPR effect to target solid tumourspassively.

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2.3 Active targeting

Active targeting relies on the concept molecular recognition, where the DDS is functionalised with biological moieties such as peptides, carbohydrates, antibodies, hormones (see figure 2). The targeting molecule provides site-specific targeting and delivers the therapeutic agents to the particular cell type or tissue in the body. This approach is highly desired in cancer therapy, where the anticancer agent can be selectively carried to cancer cells. In contrast to healthy cells, cancer cells express certain proteins or receptors, which the targeting molecule can specifically bind to with high affinity. This results in a highly controlled release of the drug locally with optimal therapeutic concentration without negative effects on non-target tissue.[16, 21]

Active targeting can also be utilised in diagnosis of various diseases. The DDS can be conjugated with different biomarkers for medical imaging of different diseases. The nanoparticles, which expresses high number of functional groups, can be conjugated with multiple imaging probes which improves the imaging sensitivity, resolution and provide with more accurate information regarding disease status and progression. Additionally, it is also possible to conjugate both imaging probes and targeting moieties to the nanoparticle at the same time. This results in both imaging and therapy.[3, 6]

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2.4 Requirements of carrier in DDS

A DDS system can be described by two components, the therapeutic drug itself and the carrier. In order to design and formulate a successful DDS some requirements need to be fulfilled. The carrier needs to be biocompatible, non-immunogenic and biodegradable when used in vivo. The material should not evoke an immune response such as opsonisation or phagocytosis and be eliminated from the body. It should not be toxic or harm the tissues in any way. It is also beneficial to use a biodegradable polymer as carrier of the DDS. When the material has fulfilled its intended purpose degradation takes place within an acceptable period of time. Common degradable polymers used for DDS applications are polyanhydrides, polyesters and polylactic acid.[22, 23]

2.5 Polymeric nanoparticles (PNPs)

Polymeric nanoparticles (PNPs) are small particles with a size between 1-500 nm, which have received a lot of attention as novel DDS used for treatment of cancer, infection, inflammation and other diseases. PNPs are the ideal DDS because of its properties as small size, high surface area, and its flexibility in surface characteristics due to simplicity of modifications and functionalisation. [10, 24, 25]

The size of PNPs in the nanometre range contributes with the ability to pass different biological barriers such as cell membranes, and cellular compartments such as nucleus.[8]_The high surface area facilitates increased loading capacity of therapeutic agents, probes and proteins. In addition, the high number of displayed functional groups can be used to attach different biological moieties as peptides, carbohydrates or antibodies for site-specific targeting. It is also possible to design a nanoparticle with multiple properties by the combination of attachment of targeting molecules and imaging probes. This results in the ability to use the nanoparticle in both therapeutic and diagnostic purposes of various diseases at the same time. It is also possible to formulate the nanoparticle with a labile linkage between drug and chain in the interior, which can respond to various external stimuli such as pH, ionic strength or temperature. This initiates a regulated release of drug at target site with optimal therapeutic concentration of the drug. [6, 24]

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2.6 Self-assembly and PEGylation

PNPs are mainly made of amphiphilic polymers. An amphiphilic polymer is a block copolymer, consisting of a hydrophilic and a hydrophobic segment. In aqueous environments these chains self-assembly above the critical micelles concentration (CMC) due to hydrophobic interactions. The self-assembled chains form particles with a core-shell morphology. The core of the particle is hydrophobic and the shell is hydrophilic. The therapeutic drug or proteins can be either physically encapsulated (hydrophobic interactions, electrostatic interactions) or via covalent interactions.[10, 26]

The therapeutic index is low for many anticancer drugs due to their low aqueous solubility, short blood circulation time, broad distribution and rapid clearance via the reticuloendothelial system(RES) system. PEGylation is a common approach used in drug delivery, where the nanoparticle is grafted with polyethylene glycol(PEG) chains in order to increase hydrodynamic volume, water solubility and biocompatibility of the particle. The attached PEG chains protects the particle from recognition by the immune system, increases the size of the particles and increases the hydrophilicity. All of this results in an enhanced EPR-effect and increased accumulation at tumor site.[6]

2.7 Dendritic polymers

Dendritic polymers have gained a lot of attention as building blocks of drug carriers because of its unique set of properties as their globular shape, high degree of branching and high number of functional groups. They can be used in biomedical applications such as drug delivery, diagnosis and gene delivery. [27]

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2.8 hb LD- hybrids

A hb LD-hybrid material consists of a linear block covalently linked to an irregularly branched dendron. hb LD-hybrid materials are synthesized via the one-step Fischer esterification reaction of ABx monomers (x ≥ 2). The A monomer (linear chain) is covalently attached to the B monomer, which have 2 active sites. This results in a material with a central core bound to a layer-by-layer structure. Each added layer is detonated as generation.[28]_{The linear chain} consists mostly of PEG due to its beneficial properties as non-immunogenicity, biocompatibility and hydrophilicity.[31, 32]_{Bis-MPA is used as building block of the dendron due} to its inherent properties as biodegradability, non-immunogenicity and biocompatibility.[33] A hyperbranched structure can be described by three repeating units; linear(L), dendritic(D) and terminal(T). According a work performed by Magnusson et al.[25]_{the units give rise to} different chemical shifts in 13_{C-NMR (400 MHz, DMSO-D6), which allows characterization of} the bis-MPA based material. Hyperbranched polymers based of bis-MPA have chemical shifts: 50.1 for terminal, 48.2 for linear, 46.2 for dendritic and 49.4 for unreacted bis-MPA.[25]

2.9 Dendritic nanogel

A nanogel is defined as a chemically cross-linked polymeric micelle, also called unimolecular micelle.[34, 35]_{Nanogels are mentioned as the ideal drug delivery system because of its} properties as high drug loading capacity, mechanical stability and ability to response to various environmental stimuli such as pH, temperature and ionic strength.[34]_{These properties} gives a delivery system, which is specific and has a highly controlled release profile of the drug. The drug concentration is below the limit which would cause toxic side effects and above the limit for proper therapeutic effect.[36]

A dendritic nanogel is a cross-linked micelle made of hb LD- hybrid chains. A key issue with intravenous administration of drug loaded micelles is disassembly of chains when reaching concentrations below CMC due to dilution effect or interaction with biological molecules in the body. This results in pre-release of cargo at non-target site and accumulation of suboptimal concentrations of therapeutic agent at target site. In order to overcome this issue cross-linking is performed to increase stability of the micelle in vivo.[6, 7]

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2.10 Purpose of project

The main purpose with this project was to study if hb LD-hybrid materials could be used as material for fabrication of dendritic nanogels used as potential polymeric DDS. A library of hb LD-hybrids was synthesized from generation two to four with different molecular weights (2kDa and 5kDa) of the linear hydrophilic mPEG chain.

The project was divided in 6 sub tasks:

 Fabrication of 2,2-bismethylolpropionic acid(bis-MPA) based hb LD- hybrids with different generations(G2-G4) via the Fischer esterification reaction. Two different lengths of the hydrophilic mPEG chain were used, 2 kDa and 5 kDa.

 Post functionalisation of the terminal hydroxyl groups of hb mPEG-nK-Gm-OH with hydrophobic allyl groups. This resulted in a material with surface reactive groups for further conjugation with designed functional groups and cross-linking of the chains.

 Tested the synthesized materials quality of micelle formation in water and selected the ones with the best ability for fabrication of dendritic nanogels. The micelle formation ability and the hydrodynamic volume of the assembled micelles were characterized with the Dynamic light scattering (DLS) equipment.

 UV-induced cross-linking of the hydrophobic core of the selected materials to fabricate dendritic nanogels. The amount of cross-linker was varied.

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3. Experimental Section

3.1 Materials

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3.2 Characterization methods

3.2.1. Nuclear magnetic resonance(NMR)

The synthesized materials (hb nK-Gm-OH) and the functionalised materials (hb mPEG-nk-Gm-allyl) were characterized using 1_{H-NMR and}13_C-NMR.1_{H-NMR and}13_{C -NMR for} structure analysis were recorded on a Bruker Advance instrument using CDCl3 and DMSO-d6 as solvent. 1_{H-NMR (400 MHz) were acquired using a spectral window of 20 ppm, a relaxation} delay of 1 second and 16 scans. 1_{H-NMR spectroscopy was performed by dissolving 10-20 mg} of sample in 1 mL of CDCl3-D6. 13_{C-NMR (100 MHz) spectra were acquired using a spectral} window of 239 ppm, a relaxation delay of 2 sec and 512 scans.13 _{C-NMR spectroscopy was} performed by dissolving 100 mg of sample in 1 mL of DMSO-D6.. Quantitative 13_{C-NMR was} performed by dissolving 150 mg of sample and 15 mg of Cr (3) in 1 mL of DMSO-D6. An inverse gated decoupling method was used with 2048 scans and a relaxation time of 5 sec.

3.2.2. Dynamic light scattering(DLS)

The DLS equipment was used to study the self-assembly ability of the synthesized materials and the hydrodynamic size of the formed micelles and the fabricated dendritic nanogels. DLS measurements were conducted with a Malvern Zetasizer NanoZS at 37 ℃, using deionized water or THF as solvent. Three different concentrations of material (5 mg/mL, 2.5 mg/mL and 0.5 mg/mL) were prepared and analysed. Each sample was allowed to equilibrate for 2 min at measurement temperature prior to analysis. Each sample was recorded 3 times, each consisting of 10 runs.

3.2.3. IR-spectroscopy

IR analyses were performed on a PerkinElmer Spectrum 100 FTIR equipped with a heat controlled single reflection attenuated total reflection(ATR) accessory (Golden Gate heat controlled) from Specac Ltd. Samples were analysed from a starting wavelength of 600 to 4000 cm-1_{. A total number of 16 scans were performed for each sample with a resolution of} 4 cm-1_{. The background normalization was performed between the wavelengths of 600 and} 4000 cm-1 _{using the average of 16 scans. Normalization was performed against the CH2} bending absorbance from the PEG core found at 1445 cm-1_.

3.2.4. UV irradiation

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3.2.5. Size Exclusion Chromatography (SEC)

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3.3. Synthetic procedures

3.3.1 General procedure of synthesis of hb LD- hybrid materials

The hb LD-hybrid materials were synthesized via the Fisher esterification reaction, generation 2 to 4 with two different lengths of the mPEG chain (2kDa & 5kDa). To a two-necked round- bottom flask, equipped with a distillation condenser, magnetic stirrer and an inlet for nitrogen gas, mPEG was added and heated to 130 ℃. Once the mPEG had melted the water flush was initiated in combination with nitrogen supply for approx. 10 min. Bis-MPA and p-TSA (acid catalyst) were then added and the reaction proceeded for 60 min under nitrogen flushing (see figure 3). Every 60 min another generation of bis-MPA and p-TSA was added. This step was repeated until desired pseudo-generation was obtained (the exact amount of reactants for synthesis of each material are described below). When the desired generation had been reached additionally 60 min nitrogen flush was applied. The nitrogen flush was turned off and the reaction proceeded overnight (18 h) under vacuum supply.Vacuum supply is essential in order to push the reaction towards completion.

The obtained viscous melt was then poured out from the reaction vessel and precipitated in a beaker containing diethyl ether. Vacuum filtration was performed and the material was collected as a white solid and put under vacuum overnight in order to evaporate remaining CHCl3. The materials were characterised by IR-spectroscopy, 13_{C-NMR and}1_H-NMR.

Figure 3: shows the general synthesis scheme of the fabricated hb LD- hybrid materials, where bis-MPA reacts with mPEG at 130 ◦ C and p-TSA is used as a catalyst.

Synthesis of mPEG-5k-G2-OH

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13 (92 %, 18.4 g). See 1_{H-NMR spectra in figure S1,}13_{C-NMR spectra in figure S13 & S16 in} supportive information.

The material was generated according the general synthesis procedure between bis-MPA and mPEG. mPEG (20 g, 4 mmol), bis-MPA (31 equiv, 124 mmol 16,62 g) and p-TSA (5 wt %, 837 mg) added to the reaction vessel. The product was collected as a white powder (87 %, 17.6 g). See 1_{H-NMR spectra in figure S2,}13_{C-NMR spectra in figure S13 & S16 for the} material in supportive information.

The material was generated according the general synthesis procedure between bis-MPA and mPEG. mPEG (20 g, 4 mmol), bis-MPA (15 equiv, 60 mmol 8.04 g) and p-TSA (5 wt %, 405 mg) added to the reaction vessel. The product was collected as a white powder (97 %, 19.3 g). See 1_{H-NMR spectra in figure S3,}13_{C-NMR spectra in figure S13 & S16 for the} material in supportive information.

The material was generated according the general synthesis procedure between bis-MPA and mPEG. mPEG (20 g, 4 mmol), bis-MPA (3 equiv, 30 mmol, 4.02 g) and p-TSA (5 wt %, 201 mg) added to the reaction vessel. The product was collected as a white powder (81 %, 16.2 g). See 1_{H-NMR spectra in figure S4,}13_{C-NMR spectra in figure S14 & S15 for the} material in supportive information.

The material was generated according the general synthesis procedure between bis-MPA and mPEG. mPEG (20 g, 4 mmol), bis-MPA (7 equiv, 70 mmol, 9.38 g) and p-TSA (5 wt %, 469 mg) added to the reaction vessel. The product was collected as a white powder (99.7 %, 19.9 g). See 1_{H-NMR spectra in figure S5,}13_{C-NMR spectra in figure S14 & S15 for the} material in supportive information.

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3.3.2 General procedure of CDI activated functionalisation of hb

mPEG-nK-Gm-OH materials

Functionalisation of all generated hb mPEG-nK-Gm-OH materials was performed with hydrophobic allyl groups in order to be able to crosslink them to form nanogels and further conjugate them with different functional groups. 3 grams of each material was functionalised with allyl groups.

CDI, 4-pentanoic acid and chloroform were added to a 50 mL round bottle flask, sealed with a septum and put on magnetic stirring for 1 h in room temperature. Activated acid was formed along with imidazole and CO2 (see step 1 in figure 4). To conform the activation of the acid 1_{H-NMR was performed. hb mPEG-nK-Gm-OH and the catalyst CsF were then added to the} reaction vessel and heated to 45 ℃ (step 2 in figure 4).The reaction was allowed to proceed under stirring overnight.

1_{H-NMR and}13_{C-NMR were performed next day in order to monitor the reaction. Remains of} activated acid was quenched with water. The product was then isolated by extraction with aqueous NaHSO4 (10 wt %) and NaHCO3 (10 wt %). The product was firstly extracted 2 times with NaHSO4 to remove imidazole and then 2 times with NaHCO3 to remove residual acid. MgSO4 was then added to remove the water. The CHCl3 was evaporated and the product was put on vacuum. The product was collected as a white/yellow solid. See used amount of reactants for synthesis of each material below.

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Synthesis of mPEG-5k-G2-allyl

CDI (6.22 mmol, 1.007 g), 4-pentanoic acid (1.5 equiv/OH, 6.22 mmol, 0.61 mL) and chloroform (1 M of 4-pentenoic acid, 6.2 mL) were added to a 50 mL round bottle flask. The reaction proceeded until activation had been completed. mPEG-5k-G2-OH (0.516 mmol, 3 g), CsF (0.2 equiv/OH, 0.126 g) was added to the reaction vessel. The temperature was set to 45 ℃ and the reaction proceeded overnight. The product was collected as white solid (1.6 g, 53 %). SEC (Mw= 56 817 g/mol, D=1.03). See 1_{H-NMR spectra in figure S7 and}13_{C-NMR spectra} in figure S18 for the material in supportive information.

CDI (10.69 mmol, 1.737 g), 4-pentanoic acid (1.5 equiv/OH, 10.69 mmol, 1.05 mL) and chloroform (1 M of 4-pentenoic acid, 10.7 mL) were added to a 50 mL round bottle flask. The reaction proceeded until activation had been completed. mPEG-5k-G3-OH (0.445 mmol, 3 g), CsF (0.2 equiv/OH, 0.217 g) was added to the reaction vessel. The temperature was set to 45 ℃ and the reaction continued overnight. The product was collected as white solid (0.94 g, 31%). SEC (Mw= 52 154 g/mol, D=1.16). See 1_{H-NMR spectra in figure S8 and}13_C-NMR spectra in figure S18 for the material in supportive information.

CDI (6.20 mmol, 1.007g), 4-pentanoic acid (1.5 equiv/OH, 6.20mmol, 0.61 mL) and chloroform (1 M of 4-pentenoic acid, 10.7 ml) were added to a 50 mL round bottle flask. The reaction proceeded until activation had been completed. mPEG-5k-G3-OH (0.517 mmol, 3 g), CsF (0.2 equiv/OH, 0.827 g) was added to the reaction vessel. The temperature was set to 45 ℃ and the reaction proceeded overnight. The product was collected as white solid (1.4 g, 55 %). SEC (Mw= 49 210 g/mol, D=1.62). See 1_{H-NMR spectra in figure S9 and}13_C-NMR spectra in figure S18 for the material in supportive information.

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CDI (13.2 mmol, 2.15 g), 4-pentanoic acid (1.5 equiv/OH, 13.2 mmol, 1.3 mL) and chloroform (1 M of 4-pentenoic acid, 13.2mL) were added to a 50 mL round bottle flask. The reaction proceeded until activation had been completed. mPEG-2k-G3-OH (1.1 mmol, 3 g), CsF (0.2 equiv/OH, 0.267 g) were then added to the reaction vessel. The temperature was set to 45 ℃ and the reaction proceeded overnight. The product was collected as a white solid (2.18 g, 73%). SEC (Mw= 24 660 g/mol, D=1.11). See 1_{H-NMR spectra in figure S11 and}13 C-NMR spectra in figure S17 for the material in supportive information.

CDI (19.3 mmol, 3.13 g), 4-pentanoic acid (1.5 equiv/OH, 6.20mmol, 1.9 mL) and chloroform (1 M of 4-pentenoic acid, 19.3 mL) were added to a 50 mL round bottle flask. The reaction proceeded until activation had been completed. mPEG-2k-G3-OH (0.802 mmol, 3 g), CsF (0.2 equiv/OH, 0.390 g) were then added to the reaction vessel. The temperature was set to 45 ℃ and the reaction proceeded overnight. The product was collected as a white solid (2.2 g, 72 %).SEC (Mw= 32 095 g/mol, D=1.27). See 1_{H-NMR spectra in figure S12 and}13_C-NMR spectra in figure S17 for the material in supportive information.

3.4 Self- assembly in water

The six fabricated hb mPEG-nk-Gm-allyl materials were prepared in the concentrations 0.5 mg/mL, 2.5 mg/mL and 5.0 mg/mL in order to study their ability to self-assembly into micelles in H2O. Each hb mPEG-nk-Gm material was dissolved in 200 μL CHCl3. The chloroform was allowed to evaporate by the supply of nitrogen gas for 2 min followed by vacuum for 1 min in combination with slow rotation of the vial. A thin film of material was formed on the wall of the vial. 2 mL deionized water filtered through a 0.2 μm syringe filter was added to each vial and the samples were vortexed for 25 sec. The samples were then put in ultrasonic bath for 20 min. The micelle formation ability and hydrodynamic volume were studied with the DLS equipment. See results for the obtained hydrodynamic volume of the micelle for each material.

3.5 Dendritic nanogel formation

In order to produce dendritic nanogels of the fabricated hb 2k-G2-allyl and hb mPEG-5k-G4-allyl materials varying amounts of the cross-linker trimetylolpropane tris (3-mercaptopropionate) were used. The dendritic nanogels were prepared at the concentration 5 mg/mL with a total volume of 10 mL.

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17 conjugate with many functional groups. In order to be able to conjugate the residual allyl groups with higher number of functional groups minimum number allyl groups were selected for cross-linking. The number of allyl groups used for cross-linking can be find in table 1 for

hb mPEG-5k-G4-allyl and in table 2 for hb mPEG-2k-G2-allyl. The amount of cross-linker was

calculated according equation 1 below (data for each material can be find in tables S1-S2 in supportive information). The amount of cross-linker used for cross-linking of the materials can be find in table 1 and 2 below.

𝑚𝑐𝑟𝑜𝑠𝑠−𝑙𝑖𝑛𝑘𝑒𝑟 = 𝑚𝑥 𝑀𝑥 * 𝑅𝑥 𝑅𝑡𝑜𝑡𝑎𝑙 *𝑅𝑡𝑜𝑡𝑎𝑙 *𝑀𝑦∗ Ry ( 1 3) (1) 𝑚𝑥

𝑀𝑥 = amount of hb mPEG-nk-Gm-allyl used 𝑅𝑥

𝑅𝑡𝑜𝑡𝑎𝑙 = fraction allyl groups used for cross-linking 𝑅𝑡𝑜𝑡𝑎𝑙 = total number of allyl groups

𝑀_𝑦 = Molecular weight of cross-linker Ry = Degree of functionality of cross-linker

Table 1: mass of cross-linker used for fabrication of dendritic nanogels based of hb mPEG-5k-G4-allyl.

hb mPEG-5k-G4-allyl 𝐦_{𝐜𝐫𝐨𝐬𝐬−𝐥𝐢𝐧𝐤𝐞𝐫} Volume cross-linker

(μL)

Ratio

Dendritic nanogel 1 0.833 mg 163 0.31

Table 2: mass of cross-linker used for fabrication of dendritic nanogels based of hb mPEG-2k-G2-allyl.

hb mPEG-2k-G2-allyl 𝐦𝐜𝐫𝐨𝐬𝐬−𝐥𝐢𝐧𝐤𝐞𝐫 Volume cross-linker

(μL)

Ratio

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18 was also added to each sample and the vials were covered with folie to protect from light. The samples were introduced to nitrogen gas and then vacuum in order evaporate the chloroform. A thin film of material was seen at the wall of the vial. 10 mL deionised H20 was added to each sample and they were vortexed for 25 sec. The samples were then put in ultrasonic bath for 20 minutes. The prepared materials were then exposed to UV light for 1 h for cross-linking. The formed dendritic nanogels were then studied with the DLS equipment. A few drops of respective nanogel was also added in 2 mL THF in order to confirm that the fabricated nanogels did not break when exposing them to another environment.

3.6 Functionalisation of dendritic nanogels

The fabricated dendritic nanogels with varying amount of cross-linker were functionalised with three different functional groups; anionic, cationic and hydrophobic groups (see table 3 below). Each nanogel was functionalised with each functional group respectively. The amount of functional group added to the dendritic nanogel was calculated according equation 2 below. The data for each material used for calculations can be find in table 7-8 in supportive information. The amount of respective functional group can be find in table 17-22 in supportive information.

Table 3 : the used functional groups for functionalisation of the dendritic nanogels and their molecular weights.

Functional group Name Molecular weight(Mw) g/mol

Hydrophobic 2-aminoethanethiol 77.15 Anionic 2-Mercatopropionic acid 106.14 Cationic Dodecanthiol-(1) 202.41 𝑚_{𝑓𝑢𝑛𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑔𝑟𝑜𝑢𝑝} = 𝑚𝑥 𝑀𝑥 * 𝑅𝑥 𝑅𝑡𝑜𝑡𝑎𝑙 *𝑅𝑡𝑜𝑡𝑎𝑙 *𝑀𝑦 (2) 𝑚𝑥

𝑀𝑥 = amount of hb mPEG-nk-Gm-allyl used 𝑅𝑥

𝑅𝑡𝑜𝑡𝑎𝑙 = fraction allyl groups used for functionalisation 𝑅𝑡𝑜𝑡𝑎𝑙 = total number of allyl groups

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19 In total 12 dendritic nanogels were functionalised; 4 with cationic groups, 4 with anionic groups and 4 with hydrophobic groups.

The calculated volume of functional group was taken from the prepared stock solution and added to a vial containing 2.5 mL dendritic nanogel. 200 μL initiator was added to each vial. The volume was adjusted with 20-25 % THF. The vials were vortexed for 25 sec and put in ultrasonic bath for 20 minutes. The vials were then exposed to UV-light for 1 h.

After functionalisation purification was performed using dialysis. The dendritic nanogels were put in dialysis membranes, which were then put in beakers containing H20/THF (90:10). They were put under magnetic stirring for 2 h. The solution was then exchanged to water and the dialysis was performed overnight. Next day the water was exchanged 5 times every 1 hour. The membranes containing the nanogels were then transferred to plastic tubes.

1 mL of six of the functionalised dendritic nanogel was taken from the tubes, added to vials and freeze dried overnight. Next day 1 mL CDCl3 was added to each vial and 1_{H-NMR was} performed. 1_{H-NMR was performed in order to confirm that functionalisation of the nanogels} were successfully made.

DLS measurements were also performed to confirm stability of the nanogel after functionalisation and determination of the hydrodynamic volume of the nanogels. Each nanogel was diluted with water to the concentration 1 mg/mL.

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20

4. Results and Discussion

4.1 Synthesis of hb mPEG-nk-Gm-OH materials

In total six hb LD- hybrid materials were synthesized successfully, generation 2-4 with two different lengths of the mPEG block, 2 kDa and 5 kDa. The fabricated materials can be found in table 4 below.

Table 4: the table provides with information of the fabricated materials such as molecular weight, architecture, size of mPEG chain, molecular weight(Mw) and end-group.

Characterization of the generated hb LD-hybrid materials were conducted by 13_C-NMR, 1_{H-NMR and IR-spectrometry.}

In the 13_{C-NMR spectra for all the materials two regions of the spectra were studied, the} carbonyl shifts in the region of 172-175 ppm, and the presence of the three distinct peaks in the region 55-45 ppm (see figure 6 below). These peaks have chemical shifts (δ): 50.7 (T), 48.7(L) and 46.7 (D) ppm. T stands for terminal, L stands for linear and D stands for dendritic units of the hybrid material (see structure in figure 6 below). [25]

As can also be seen in figure 6 below, the absence of a peak around 178 ppm indicates no free R´COOH left, meaning that all bis-MPA had reacted. Distinct peaks between 172-174 ppm corresponds to ester groups in the dendron part of the hb LD-material, which indicates that desired hb LD-hybrid material was fabricated. The 13_{C-NMR spectra for all fabricated materials} can be find in figures S13-S16 in supportive information.

Name Architecture mPEG(Da) Generation Mw(g/mol) End- group

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21

Figure 6: 13C-NMR spectra of hb mPEG-2k-G3-OH with zoom of the region 180-170 ppm and 55-45 ppm,

and the structure of hb mPEG-2k-G3-OH labelled with the different possible units: linear(L), dendritic(D) and terminal(T).

As can be seen in the 1_{H-NMR spectra in figure 7 below four distinct peaks with different} chemical shifts were studied to characterize that desired hb mPEG-nK-Gm-OH was fabricated. The peaks had chemical shifts (ᵹ): 1.14 ppm equal to –CH3, 3,65 ppm equal to CH2-CH2-O (mPEG), 3.39 ppm equal to –CH2-OC-O (bis-MPA). All the six synthesized hb mPEG-nK-Gm-OH materials were identified by this approach. The integrals for all generations confirmed that the desired product had been synthesized. The 1_{H-NMR spectra for all six synthesized} materials with integrals can be found in supportive information.

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22 The IR-spectra in figures 8-9 below shows the absence of any free carboxylic groups, which confirms that all residual bis-MPA have attached to the mPEG chain. The carboxylic stretching at 1683 cm-1_{is shifted to the carbonyl stretching at 1727 cm}-1_{for all hb mPEG-nk-Gm-OH} materials, which indicates that the reaction is completed and desired materials are generated. One can also see an increase in ester (R-COOR´) bonds with increased generation due to more attached bis-MPA units.

Figure 8: IR-spectra of hb mPEG-2k-Gm-OH (G2-G4) showing shift from 1683 cm-1 _{(free bis-MPA) to 1727 cm}-1 corresponding to ester bond in the dendron part of the hb LD- hybrid material.

Figure 9: IR-spectra of hb mPEG-5k-Gm-OH (G2-G4) showing shift from 1683 cm-1 _{(free bis-MPA) to 1727 cm}-1

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23

4.2 CDI activated functionalisation of hb mPEG-nk-Gm-OH with allyl groups

Functionalisation with hydrophobic allyl groups was successful for all materials. The 1_H-NMR spectra in figure 10 confirms conversion of OH functionality to allyl functionality. The conversion of functionality was confirmed by presence of two distinct peaks between 5-6 ppm in the 1_{H-NMR spectra for mPEG-2k-G3-allyl in comparison to the}1_{H-NMR for} mPEG-2k-G3-allyl in figure 10 below.

Figure 10: (a): 1_{H-NMR for mPEG-2k-G3-OH showing no peaks for allyl groups. (b):}1_{H-NMR for}

mPEG-2k-G3-allyl where full conversion of hydroxyl functionalities to allyl functionalities is confirmed.

4.3 Micelle formation ability

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24 The materials with the best micelle formation ability were mPEG-5k-G4-allyl and mPEG-2k-G2-allyl. See figure 11-12 for the DLS measurements at the concentration 2.5 mg/mL for both materials below. These materials ratio between hydrophilic and hydrophobic segments resulted in best self-assembly (table 5) and were chosen as candidates for nanogel fabrication and further functionalisation of the inner core with ligands as cationic, anionic and hydrophobic groups.

Table 5: shows the six hb mPEG-nK-Gm-allyl materials and their ratio between hydrophilic and hydrophobic segments.

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25 Figure 12: the result from the DLS measurements at concentration 2.5 mg/mL showing the micelle formation ability of mPEG-5k-G4-allyl in H20. Z-average:127.9 nm, PDI= 0.235

4.4 Fabrication of dendritic nanogels

Four dendritic nanogels were fabricated in total, two made of mPEG-2k-G2-allyl and two made of mPEG-5k-G4-allyl. For each material two different degree of cross-linking were used. The amount of cross-linker used for formation of dendritic nanogels of mPEG-2k-G2-allyl were 25 % respectively 75 %. For fabrication of dendritic nanogels based on mPEG-5k-G4 63 % respectively 80 % cross-linker were used. The degree of cross-linker used for formation of dendritic nanogels resulted in nanogels with hydrodynamic volumes between 147-200 nm. See table 6-7 below for more detailed information for respective dendritic nanogel. 100 μL of each dendritic nanogel was added in 1 mL THF respectively. The fabricated dendritic nanogels were still stable, which indicated that the cross-linking was successful for each dendritic nanogel.

Table 6: the hydrodynamic volume(nm) of fabricated dendritic nanogels made of hb mPEG-2k-G2-allyl at different degree of cross-linking.

hb mPEG-2k-G2-allyl Cross-linking (%) Z-average(nm) PDI

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26 Table 7: the hydrodynamic volume of the dendritic nanogels made of hb mPEG-5k-G4-allyl

of at different degree of cross-linking.

hb mPEG-5k-G4-allyl Cross-linking (%) Z-average(nm) PDI

Dendritic nanogel 3 31 147.4 0.391 Dendritic nanogel 4 62.5 191.4 0.377 Dendritic nanogel 3 + THF 31 151.4 0.237 Dendritic nanogel 4 + THF 62.5 204.1 0.263

4.5 Functionalisation of the inner core with different ligands

The inner core of the dendritic nanogels was functionalised with cationic, anionic and hydrophobic groups. The 1_{H-NMR spectra of six functionalised dendritic nanogels showed no} presence of peaks with chemical shifts 5-6 ppm corresponding to allyl groups (see figure 13 below). This confirmed that the functionalisation was successfully performed of all functional groups.

Figure 13: shows the 1_{H-NMR spectras of the nanogels after functionalisation with cationic, anionic}

and hydrophobic groups. The 1_{H-NMR spectras for the nanogels show no presence of peaks with}

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27 DLS measurements were made after functionalisation in order to estimate the hydrodynamic volume of the nanogel after functionalisation and confirm that the cross-linked nanogels were still intact. Table 8 below summarises the hydrodynamic volume of the functionalised nanogels. The hydrodynamic volume was below 200 nm for most of the nanogels. For some nanogels the size exceeded 200 nm, which can be because of interference of bigger particles when measured. The results indicate a smaller hydrodynamic volume with increased degree of cross-linker.

Table 8: summarises the hydrodynamic volume of the nanogels after functionalisation. The symbol * stands for potential interference of bigger particles during measurement. Filtration was not

performed.

Functional group

Nanogel Z-average PDI

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28

5. Conclusions

 A library of hb mPEG-nk-Gm-OH materials was successfully synthesized via the Fischer esterification reaction. The materials consisted of a hydrophobic core (bis-MPA) and a hydrophilic linear chain(mPEG). In total six materials were synthesized with two different lengths of the hydrophilic mPEG chain (2 kDa & 5 kDa) and with different generations, G2-G4.

 All the synthesized hb mPEG-nk-Gm-OH materials were successfully post functionalised with allyl groups via CDI mediated functionalisation.

 The materials had ability to self-assembly in water and form micelles with hydrodynamic volumes within the range 10-200 nm.

 Two materials, mPEG-2k-G2-allyl and mPEG-5k-G4-allyl were chosen as candidates for crosslinking, resulting in successful fabrication of dendritic nanogels.

 The fabricated dendritic nanogels could be conjugated with hydrophobic, cationic and anionic functional groups via the residual allyl groups in the hydrophobic inner core of the nanogel. This means that the dendritic nanogels have the ability to encapsulate proteins and hydrophobic drugs such as anticancer drug Doxorubicin(DOX).

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6. Future work

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Dendritic nanogel for drug delivery platform